Date

7-2014

Document Type

Dissertation

Degree Name

Ph.D.

Institution

Oregon Health & Science University

Abstract

The cAMP dependent protein kinase, or protein kinase A (PKA) is an important and ubiquitous intracellular mediator of information transfer in neurons, where it plays critical roles in neurotransmission, cellular excitability, and synaptic plasticity. All of these processes require high specificity in PKA phosphorylation. An abundance of studies have shown that PKA specificity is mediated by a class of proteins called A-kinase anchoring proteins (AKAPs), which target upstream activators, downstream substrates, and inhibitors of PKA to defined subcellular locations, putatively establishing tight gradients of PKA phosphorylation. However, AKAPs directly anchor only the regulatory subunit of PKA. Once bound by cAMP, conformational changes in the PKA holoenzyme result in the release of catalytic subunit of PKA (PKA-cat) whereupon it is free to diffuse. A cytosolic protein of comparable size to PKA-cat is expected to fully sample synaptic compartments, such as dendritic spines, in tens of milliseconds and exit these compartments within hundreds of milliseconds. Considering that these time scales are significantly shorter than both the enzymatic turnover of PKA-cat and the observed time scales of PKA signaling, such diffusion would be expected to break down the PKA specificity established by KAPs. Here, we use a variety of imaging and biochemical techniques in both neurons and model cell lines to investigate gradients of PKA phosphorylation and the mechanisms by which they are enhanced in both resting and stimulated conditions. Collectively, the results paint a picture in which both states of PKA phosphorylation preferentially occur in the plasma membrane compartment of cells. In resting states, PKA phosphorylation is largely confined to the plasma membrane. Similar to resting PKA phosphorylation, exogenous stimulation of the PKA holoenzyme also results in a gradient of PKA phosphorylation emanating from the plasma membrane. Furthermore, we have discovered a role for Nterminal myristoylation of PKA-cat in imparting membrane affinity to the freed PKA-cat thereby limiting its mobility and enhancing its phosphorylation in the plasma membrane. Such targeting of PKA-cat is a novel mechanism for maintaining AKAP established PKA signaling gradients. As PKA is one of the many promiscuous, yet specific, diffusible proteins involved in signal transduction, we hope that these results will provide a better template from which to understand the general principles of specificity in neuronal regulatory signaling.

Identifier

doi:10.6083/M40V8BHB

Division

Neuroscience Graduate Program

School

School of Medicine

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